A commercial assay (Amplified Mycobacterium tuberculosis Direct Test, Gen Probe) which combines transcription-mediated amplification of target rRNA with amplicon detection by a chemiluminescent DNA probe for the rapid detection of Mycobacterium tuberculosis in sputum was evaluated. The test was applied to consecutively collected, NALC/NaOH processed sputum sediments from two laboratories (H and L), each serving a different population of patients with pulmonary tuberculosis. Results were compared to those of fluorochrome staining and culture. A total of 760 specimens obtained from 246 patients were used for the study. The test was positive in 141 of 144 (98%) specimens that were fluorochrome-positive and culture-positive for Mycobacterium tuberculosis. Fifteen of 31 specimens that were fluorochrome-negative, culture-positive were also assay-positive. A total of 312 specimens (100 patients) from laboratory H (prevalence = 10%) and 448 specimens (146 patients) from laboratory L (prevalence = 34%) were analyzed. Compared to culture, test sensitivity, specificity, positive predictive and negative predictive values were 65%, 99%, 94% and 97%, respectively, for laboratory H and 93%, 99%, 99% and 97%, respectively, for laboratory L. If the results were analyzed on the basis of at least one concordant result between the amplification assay and culture in three sputum samples per patient, then the sensitivity, specificity, positive and negative predictive values for identifying infected patients was 70%, 99%, 87% and 97%, respectively, for laboratory H, and 100%, 98%, 96% and 100%, respectively, for laboratory L.